Rearrangements initiating within the well-characterized break-point cluster region of the mixed lineage leukemia (MLL) gene on 11q23 are a hallmark of therapy-related leukemias following treatment with topoisomerase II poisons including etoposide. Hematopoietic stem cells (HSC) are believed to be the target cell for leukemia-initiating MLL rearrangement events. Although etoposide treatment is sufficient to induce readily detectable MLL rearrangements in primary human CD34+ cells, the majority of cells that gain translocations do not proliferate in culture possibly due to reduced proliferative capacity of most CD34+ cells during normal differentiation [Blood 2005;105:2124]. We characterized the impact of etoposide on primary human long-term repopulating HSC that represent only a minor portion of CD34+ cells. The proliferative capacity of HSC is dramatically increased following both a single and multiple exposures to etoposide as determined by their ability to engraft bone marrow of immune-deficient non-obese diabetic/severe combined immunodeficient mice and to initiate hematopoiesis in long-term initiating cultures. Similar to results in CD34+ cells, a significant proportion of etoposide-treated HSC-derived clones harbored stable MLL rearrangements, including duplications, inversions and translocations. These results indicate HSC are highly susceptible to etoposide-induced and potentially oncogenic rearrangements initiating within MLL, and these HSC are particularly proficient for continued long-term proliferation both in vivo and in vitro.